Many Gram-negative pathogens including the Yersinia species (Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis) use a Type III Secretion System (TTSS) for pathogenesis. The TTSS is essentially a molecular syringe that injects cytotoxic proteins into host cells. In Yersinia, the TTSS delivers at least 8 proteins known as Yops into target cells, and mutants lacking a functional TTSS are avirulent. YopK is required for proper regulation of the TTSS, but the molecular details of this regulation remain a mystery. Bioinformatic analysis of YopK does not reveal any enzymatic domains or homology to known proteins. YopK is injected into host cells by the TTSS, and our data suggest that YopK acts from within host cells to regulate the TTSS. Since little is known about YopK, the work proposed here represents a comprehensive characterization of the protein. Using a deletion analysis, we will identify domains within YopK that are important for its injection, regulation of the TTSS, and its interaction with other proteins. We will also investigate its role within host cells using a combination of biochemistry, flow cytometry, and microscopy to determine YopK localization and identify interacting proteins. Insights gained from this work will provide a foundation for future studies that will elucidate the molecular details governing YopK regulation. In summary, this study will use novel approaches to investigate a protein that is important for regulation of the TTSS, the central virulence strategy of Yersinia and many other Gram-negative pathogens. Understanding the molecular mechanisms by which these pathogens regulate the TTSS will provide us with targets for alternative broad-based therapeutic strategies. PUBLIC HEALTH RELEVANCE: Gram-negative pathogens cause a variety of diseases and present a significant strain to the US health care system. Many Gram-negatives rely on a molecular syringe to inject toxic proteins into host cells;therefore blocking the syringe could be an effective antimicrobial strategy. Our work seeks to understand regulation of the syringe, and insights gained here may be helpful in designing broad-based therapeutics for treating or preventing numerous diseases.